A giant virus infecting the amoeboflagellate Naegleria.

Giant viruses (Nucleocytoviricota) are significant lethality agents of various eukaryotic hosts. Although metagenomics indicates their ubiquitous distribution, available giant virus isolates are restricted to a very small number of protist and algal hosts. Here we report on the first viral isolate that replicates in the amoeboflagellate Naegleria. This genus comprises the notorious human pathogen Naegleria fowleri, the causative agent of the rare but fatal primary amoebic meningoencephalitis. We have elucidated the structure and infection cycle of this giant virus, Catovirus naegleriensis (a.k.a. Naegleriavirus, NiV), and show its unique adaptations to its Naegleria host using fluorescence in situ hybridization, electron microscopy, genomics, and proteomics. Naegleriavirus is only the fourth isolate of the highly diverse subfamily Klosneuvirinae, and like its relatives the NiV genome contains a large number of translation genes, but lacks transfer RNAs (tRNAs). NiV has acquired genes from its Naegleria host, which code for heat shock proteins and apoptosis inhibiting factors, presumably for host interactions. Notably, NiV infection was lethal to all Naegleria species tested, including the human pathogen N. fowleri. This study expands our experimental framework for investigating giant viruses and may help to better understand the basic biology of the human pathogen N. fowleri.

Virophages: association with human diseases and their predicted role as virus killers.

The fascinating discovery of the first giant virus, Acanthamoeba polyphaga mimivirus (APMV), belonging to the family Mimiviridae in 2008, and its associated virophage, Sputnik, have left the world of microbiology awestruck. To date, about 18 virophages have been isolated from different environmental sources. With their unique feature of resisting host cell infection and lysis by giant viruses, analogous to bacteriophage, they have been assigned under the family Lavidaviridae. Genome of T-27, icosahedral-shaped, non-enveloped virophages, consist of dsDNA encoding four proteins, namely, major capsid protein, minor capsid protein, ATPase and cysteine protease, which are essential in the formation and assembly of new virophage particles during replication. A few virophage genomes have been observed to contain additional sequences like PolB, ZnR and S3H. Another interesting characteristic of virophage is that Mimivirus lineage A is immune to infection by the Zamilon virophage through a phenomenon termed MIMIVIRE, resembling the CRISPR-Cas mechanism in bacteria. Based on the fact that giant viruses have been found in clinical samples of hospital-acquired pneumonia and rheumatoid arthritis patients, virophages have opened a novel era in the search for cures of various diseases. This article aims to study the prospective role of virophages in the future of human therapeutics.

Kinetic Analysis of Acanthamoeba castellanii Infected with Giant Viruses Quantitatively Revealed Process of Morphological and Behavioral Changes in Host Cells.

Most virus-infected cells show morphological and behavioral changes, which are called cytopathic effects. Acanthamoeba castellanii, an abundant, free-living protozoan, serves as a laboratory host for some viruses of the phylum Nucleocytoviricota-the giant viruses. Many of these viruses cause cell rounding in the later stages of infection in the host cells. Here, we show the changes that lead to cell rounding in the host cells through time-lapse microscopy and image analysis. Time-lapse movies of A. castellanii cells infected with Mimivirus shirakomae, kyotovirus, medusavirus, or Pandoravirus japonicus were generated using a phase-contrast microscope. We updated our phase-contrast-based kinetic analysis algorithm for amoebae (PKA3) and used it to analyze these time-lapse movies. Image analysis revealed that the process leading to cell rounding varies among the giant viruses; for example, M. shirakomae infection did not cause changes for some time after the infection, kyotovirus infection caused an early decrease in the number of cells with typical morphologies, and medusavirus and P. japonicus infection frequently led to the formation of intercellular bridges and rotational behavior of host cells. These results suggest that in the case of giant viruses, the putative reactions of host cells against infection and the putative strategies of virus spread are diverse. IMPORTANCE Quantitative analysis of the infection process is important for a better understanding of viral infection strategies and virus-host interactions. Here, an image analysis of the phase-contrast time-lapse movies displayed quantitative differences in the process of cytopathic effects due to the four giant viruses in Acanthamoeba castellanii, which were previously unclear. It was revealed that medusavirus and Pandoravirus japonicus infection led to the formation of a significant number of elongated particles related to intercellular bridges, emphasizing the importance of research on the interaction of viruses with host cell nuclear function. Mimivirus shirakomae infection did not cause any changes in the host cells initially, so it is thought that the infected cells can actively move and spread over a wider area, emphasizing the importance of observation in a wider area and analysis of infection efficiency. These results suggest that a kinetic analysis using the phase-contrast-based kinetic analysis algorithm for amoebae (PKA3) reveals the infection strategies of each giant virus.

Structure and physiology of giant DNA viruses.

Although giant viruses have existed for millennia and possibly exerted great evolutionary influence in their environment. Their presence has only been noticed by virologists recently with the discovery of Acanthamoeba polyphaga mimivirus in 2003. Its virion with a diameter of 500 nm and its genome larger than 1 Mpb shattered preconceived standards of what a virus is and triggered world-wide prospection studies. Thanks to these investigations many giant virus families were discovered, each with its own morphological peculiarities and genomes ranging from 0.4 to 2.5 Mpb that possibly encode more than 400 viral proteins. This review aims to present the morphological diversity, the different aspects observed in host-virus interactions during replication, as well as the techniques utilized during their investigation.

Visualizing active viral infection reveals diverse cell fates in synchronized algal bloom demise.

Marine viruses are the most abundant biological entity in the ocean and are considered as major evolutionary drivers of microbial life [C. A. Suttle, Nat. Rev. Microbiol. 5, 801-812 (2007)]. Yet, we lack quantitative approaches to assess their impact on the marine ecosystem. Here, we provide quantification of active viral infection in the bloom forming single-celled phytoplankton Emiliania huxleyi infected by the large virus EhV, using high-throughput single-molecule messenger RNA in situ hybridization (smFISH) of both virus and host transcripts. In natural samples, viral infection reached only 25% of the population despite synchronized bloom demise exposing the coexistence of infected and noninfected subpopulations. We prove that photosynthetically active cells chronically release viral particles through nonlytic infection and that viral-induced cell lysis can occur without viral release, thus challenging major assumptions regarding the life cycle of giant viruses. We could also assess active infection in cell aggregates linking viral infection and carbon export to the deep ocean [C. P. Laber et al., Nat. Microbiol. 3, 537-547 (2018)] and suggest a potential host defense strategy by enrichment of infected cells in sinking aggregates. Our approach can be applied to diverse marine microbial systems, opening a mechanistic dimension to the study of biotic interactions in the ocean.

Alohomora! What the entry mechanisms tell us about the evolution and diversification of giant viruses and their hosts.

The virosphere is fascinatingly vast and diverse, but as mandatory intracellular parasites, viral particles must reach the intracellular space to guarantee their species' permanence on the planet. While most known viruses that infect animals explore the endocytic pathway to enter the host cell, a diverse group of ancient viruses that make up the phylum Nucleocytoviricota appear to have evolved to explore new access' routes to the cell's cytoplasm. Giant viruses of amoeba take advantage of the phagocytosis process that these organisms exploit a lot, while phycodnavirus must actively break through a algal cellulose cell wall. The mechanisms of entry into the cell and the viruses themselves are diverse, varying in the steps of adhesion, entry, and uncoating. These are clues left by evolution about how these organisms shaped and were shaped by convoluting with eukaryotes.

Host Range and Coding Potential of Eukaryotic Giant Viruses.

Giant viruses are a group of eukaryotic double-stranded DNA viruses with large virion and genome size that challenged the traditional view of virus. Newly isolated strains and sequenced genomes in the last two decades have substantially advanced our knowledge of their host diversity, gene functions, and evolutionary history. Giant viruses are now known to infect hosts from all major supergroups in the eukaryotic tree of life, which predominantly comprises microbial organisms. The seven well-recognized viral clades (taxonomic families) have drastically different host range. Mimiviridae and Phycodnaviridae, both with notable intrafamilial genome variation and high abundance in environmental samples, have members that infect the most diverse eukaryotic lineages. Laboratory experiments and comparative genomics have shed light on the unprecedented functional potential of giant viruses, encoding proteins for genetic information flow, energy metabolism, synthesis of biomolecules, membrane transport, and sensing that allow for sophisticated control of intracellular conditions and cell-environment interactions. Evolutionary genomics can illuminate how current and past hosts shape viral gene repertoires, although it becomes more obscure with divergent sequences and deep phylogenies. Continued works to characterize giant viruses from marine and other environments will further contribute to our understanding of their host range, coding potential, and virus-host coevolution.

Giant viruses.

Chantal Abergel and Jean-Michel Claverie introduce giant viruses.

The Mottled Capsid of the Salmonella Giant Phage SPN3US, a Likely Maturation Intermediate with a Novel Internal Shell.

"Giant" phages have genomes of >200 kbp, confined in correspondingly large capsids whose assembly and maturation are still poorly understood. Nevertheless, the first assembly product is likely to be, as in other tailed phages, a procapsid that subsequently matures and packages the DNA. The associated transformations include the cleavage of many proteins by the phage-encoded protease, as well as the thinning and angularization of the capsid. We exploited an amber mutation in the viral protease gene of the Salmonella giant phage SPN3US, which leads to the accumulation of a population of capsids with distinctive properties. Cryo-electron micrographs reveal patterns of internal density different from those of the DNA-filled heads of virions, leading us to call them "mottled capsids". Reconstructions show an outer shell with T = 27 symmetry, an embellishment of the HK97 prototype composed of the major capsid protein, gp75, which is similar to some other giant viruses. The mottled capsid has a T = 1 inner icosahedral shell that is a complex network of loosely connected densities composed mainly of the ejection proteins gp53 and gp54. Segmentation of this inner shell indicated that a number of densities (~12 per asymmetric unit) adopt a "twisted hook" conformation. Large patches of a proteinaceous tetragonal lattice with a 67 Å repeat were also present in the cell lysate. The unexpected nature of these novel inner shell and lattice structures poses questions as to their functions in virion assembly.

Structural and Proteomic Characterization of the Initiation of Giant Virus Infection.

Since their discovery, giant viruses have expanded our understanding of the principles of virology. Due to their gargantuan size and complexity, little is known about the life cycles of these viruses. To answer outstanding questions regarding giant virus infection mechanisms, we set out to determine biomolecular conditions that promote giant virus genome release. We generated four infection intermediates in Samba virus (Mimivirus genus, lineage A) as visualized by cryoelectron microscopy (cryo-EM), cryoelectron tomography (cryo-ET), and scanning electron microscopy (SEM). Each of these four intermediates reflects similar morphology to a stage that occurs in vivo. We show that these genome release stages are conserved in other mimiviruses. Finally, we identified proteins that are released from Samba and newly discovered Tupanvirus through differential mass spectrometry. Our work revealed the molecular forces that trigger infection are conserved among disparate giant viruses. This study is also the first to identify specific proteins released during the initial stages of giant virus infection.

A virophage cross-species infection through mutant selection represses giant virus propagation, promoting host cell survival.

Virus adaptation to new hosts is a major cause of infectious disease emergence. This mechanism has been intensively studied in the context of zoonotic virus spillover, due to its impact on global health. However, it remains unclear for virophages, parasites of giant viruses and potential regulators of microbial communities. Here, we present, for the first time to our knowledge, evidence of cross-species infection of a virophage. We demonstrated that challenging the native population of Guarani virophage with two previously unidentified giant viruses, previously nonpermissive to this virophage, allows the selection of a mutant genotype able to infect these giant viruses. We were able to characterize the potential genetic determinant (deletion) carried by the virophage with the expanded-host range. Our study also highlights the relevant biological impact of this host adaptation by demonstrating that coinfection with the mixture containing the mutant virophage abolishes giant virus production and rescues the host cell population from lysis.

Virus goes viral: an educational kit for virology classes.

BACKGROUND: Viruses are the most numerous entities on Earth and have also been central to many episodes in the history of humankind. As the study of viruses progresses further and further, there are several limitations in transferring this knowledge to undergraduate and high school students. This deficiency is due to the difficulty in designing hands-on lessons that allow students to better absorb content, given limited financial resources and facilities, as well as the difficulty of exploiting viral particles, due to their small dimensions. The development of tools for teaching virology is important to encourage educators to expand on the covered topics and connect them to recent findings. Discoveries, such as giant DNA viruses, have provided an opportunity to explore aspects of viral particles in ways never seen before. Coupling these novel findings with techniques already explored by classical virology, including visualization of cytopathic effects on permissive cells, may represent a new way for teaching virology. This work aimed to develop a slide microscope kit that explores giant virus particles and some aspects of animal virus interaction with cell lines, with the goal of providing an innovative approach to virology teaching. METHODS: Slides were produced by staining, with crystal violet, purified giant viruses and BSC-40 and Vero cells infected with viruses of the genera Orthopoxvirus, Flavivirus, and Alphavirus. Slides with amoebae infected with different species of giant viruses and stained with hemacolor reagents were also produced. RESULTS: Staining of the giant viruses allowed better visualization of the viral particles, and this technique highlights the diversity in morphology and sizes among them. Hemacolor staining enabled visualization of viral factories in amoebae, and the staining of infected BSC-40 and Vero cell monolayers with crystal violet highlights plaque-forming units. CONCLUSIONS: This kit was used in practical virology classes for the Biological Sciences course (UFMG, Brazil), and it will soon be made available at a low-cost for elementary school teachers in institutions that have microscopes. We hope this tool will foster an inspiring learning environment.

Influence of light on the infection of Aureococcus anophagefferens CCMP 1984 by a "giant virus".

The pelagophyte Aureococcus anophagefferens has caused recurrent brown tide blooms along the northeast coast of the United States since the mid-1980's, and more recently spread to other regions of the globe. These blooms, due to the high cell densities, are associated with severe light attenuation that destroys the sea grass beds which provide the basis for many fisheries. Data collected by transmission electron microscopy, PCR, and metatranscriptomic studies of the blooms, support the hypothesis that large dsDNA viruses play a role in bloom dynamics. While a large (~140 nm) icosahedral virus, with a 371 kbp genome, was first isolated more than a decade ago, the constraints imposed by environmental parameters on bloom infection dynamics by Aureococcus anophagefferens Virus, (AaV) remain unknown. To investigate the role light plays in infection by this virus, we acclimated A. anophagefferens to light intensities of 30 (low), 60 (medium) or 90 µmol photons m-2 s-1 (high) and infected cultures at these irradiance levels. Moreover, we completed light shift experiments where acclimated cultures were exposed to even lower light intensities (0, 5, and 15 µmol photons m-2 s-1) consistent with irradiance found during the peak of the bloom when cell concentrations are highest. The abundance of viruses produced per lytic event (burst size) was lower in the low irradiance acclimated cultures compared to the medium and high acclimated cultures. Transferring infected cultures to more-limiting light availabilities further decreased burst size and increased the length of time it took for cultures to lyse, regardless of acclimation irradiance level. A hypothetical mechanism for the reduced efficiency of the infection cycle in low light due to ribosome biogenesis was predicted from pre-existing transcriptomes. Overall, these studies provide a framework for understanding light effects on infection dynamics over the course of the summer months when A. anophagefferens blooms occur.

Exploration of the propagation of transpovirons within Mimiviridae reveals a unique example of commensalism in the viral world.

Acanthamoeba-infecting Mimiviridae are giant viruses with dsDNA genome up to 1.5 Mb. They build viral factories in the host cytoplasm in which the nuclear-like virus-encoded functions take place. They are themselves the target of infections by 20-kb-dsDNA virophages, replicating in the giant virus factories and can also be found associated with 7-kb-DNA episomes, dubbed transpovirons. Here we isolated a virophage (Zamilon vitis) and two transpovirons respectively associated to B- and C-clade mimiviruses. We found that the virophage could transfer each transpoviron provided the host viruses were devoid of a resident transpoviron (permissive effect). If not, only the resident transpoviron originally isolated from the corresponding virus was replicated and propagated within the virophage progeny (dominance effect). Although B- and C-clade viruses devoid of transpoviron could replicate each transpoviron, they did it with a lower efficiency across clades, suggesting an ongoing process of adaptive co-evolution. We analysed the proteomes of host viruses and virophage particles in search of proteins involved in this adaptation process. This study also highlights a unique example of intricate commensalism in the viral world, where the transpoviron uses the virophage to propagate and where the Zamilon virophage and the transpoviron depend on the giant virus to replicate, without affecting its infectious cycle.

Giant virus vs amoeba: fight for supremacy.

Since the discovery of mimivirus, numerous giant viruses associated with free-living amoebae have been described. The genome of giant viruses can be more than 2.5 megabases, and virus particles can exceed the size of many bacteria. The unexpected characteristics of these viruses have made them intriguing research targets and, as a result, studies focusing on their interactions with their amoeba host have gained increased attention. Studies have shown that giant viruses can establish host-pathogen interactions, which have not been previously demonstrated, including the unprecedented interaction with a new group of small viruses, called virophages, that parasitize their viral factories. In this brief review, we present recent advances in virophage-giant virus-host interactions and highlight selected studies involving interactions between giant viruses and amoebae. These unprecedented interactions involve the giant viruses mimivirus, marseillevirus, tupanviruses and faustovirus, all of which modulate the amoeba environment, affecting both their replication and their spread to new hosts.

Targeted metagenomic recovery of four divergent viruses reveals shared and distinctive characteristics of giant viruses of marine eukaryotes.

Giant viruses have remarkable genomic repertoires-blurring the line with cellular life-and act as top-down controls of eukaryotic plankton. However, to date only six cultured giant virus genomes are available from the pelagic ocean. We used at-sea flow cytometry with staining and sorting designed to target wild predatory eukaryotes, followed by DNA sequencing and assembly, to recover novel giant viruses from the Pacific Ocean. We retrieved four 'PacV' partial genomes that range from 421 to 1605 Kb, with 13 contigs on average, including the largest marine viral genomic assembly reported to date. Phylogenetic analyses indicate that three of the new viruses span a clade with deep-branching members of giant Mimiviridae, incorporating the Cafeteria roenbergensis virus, the uncultivated terrestrial Faunusvirus, one PacV from a choanoflagellate and two PacV with unclear hosts. The fourth virus, oPacV-421, is phylogenetically related to viruses that infect haptophyte algae. About half the predicted proteins in each PacV have no matches in NCBI nr (e-value < 10-5), totalling 1735 previously unknown proteins; the closest affiliations of the other proteins were evenly distributed across eukaryotes, prokaryotes and viruses of eukaryotes. The PacVs encode many translational proteins and two encode eukaryotic-like proteins from the Rh family of the ammonium transporter superfamily, likely influencing the uptake of nitrogen during infection. cPacV-1605 encodes a microbial viral rhodopsin (VirR) and the biosynthesis pathway for the required chromophore, the second finding of a choanoflagellate-associated virus that encodes these genes. In co-collected metatranscriptomes, 85% of cPacV-1605 genes were expressed, with capsids, heat shock proteins and proteases among the most highly expressed. Based on orthologue presence-absence patterns across the PacVs and other eukaryotic viruses, we posit the observed viral groupings are connected to host lifestyles as heterotrophs or phototrophs. This article is part of a discussion meeting issue 'Single cell ecology'.

A Puzzling Anomaly in the 4-Mer Composition of the Giant Pandoravirus Genomes Reveals a Stringent New Evolutionary Selection Process.

Pandoraviridae is a rapidly growing family of giant viruses, all of which have been isolated using laboratory strains of Acanthamoeba The genomes of 10 distinct strains have been fully characterized, reaching up to 2.5 Mb in size. These double-stranded DNA genomes encode the largest of all known viral proteomes and are propagated in oblate virions that are among the largest ever described (1.2 µm long and 0.5 µm wide). The evolutionary origin of these atypical viruses is the object of numerous speculations. Applying the chaos game representation to the pandoravirus genome sequences, we discovered that the tetranucleotide (4-mer) "AGCT" is totally absent from the genomes of 2 strains (Pandoravirus dulcis and Pandoravirus quercus) and strongly underrepresented in others. Given the amazingly low probability of such an observation in the corresponding randomized sequences, we investigated its biological significance through a comprehensive study of the 4-mer compositions of all viral genomes. Our results indicate that AGCT was specifically eliminated during the evolution of the Pandoraviridae and that none of the previously proposed host-virus antagonistic relationships could explain this phenomenon. Unlike the three other families of giant viruses (Mimiviridae, Pithoviridae, and Molliviridae) infecting the same Acanthamoeba host, the pandoraviruses exhibit a puzzling genomic anomaly suggesting a highly specific DNA editing in response to a new kind of strong evolutionary pressure.IMPORTANCE Recent years have seen the discovery of several families of giant DNA viruses infecting the ubiquitous amoebozoa of the genus Acanthamoeba With double-stranded DNA (dsDNA) genomes reaching 2.5 Mb in length packaged in oblate particles the size of a bacterium, the pandoraviruses are currently the most complex and largest viruses known. In addition to their spectacular dimensions, the pandoraviruses encode the largest proportion of proteins without homologs in other organisms, which is thought to result from a de novo gene creation process. While using comparative genomics to investigate the evolutionary forces responsible for the emergence of such an unusual giant virus family, we discovered a unique bias in the tetranucleotide composition of the pandoravirus genomes that can result only from an undescribed evolutionary process not encountered in any other microorganism.

Virophages of Giant Viruses: An Update at Eleven.

The last decade has been marked by two eminent discoveries that have changed our perception of the virology field: The discovery of giant viruses and a distinct new class of viral agents that parasitize their viral factories, the virophages. Coculture and metagenomics have actively contributed to the expansion of the virophage family by isolating dozens of new members. This increase in the body of data on virophage not only revealed the diversity of the virophage group, but also the relevant ecological impact of these small viruses and their potential role in the dynamics of the microbial network. In addition, the isolation of virophages has led us to discover previously unknown features displayed by their host viruses and cells. In this review, we present an update of all the knowledge on the isolation, biology, genomics, and morphological features of the virophages, a decade after the discovery of their first member, the Sputnik virophage. We discuss their parasitic lifestyle as bona fide viruses of the giant virus factories, genetic parasites of their genomes, and then their role as a key component or target for some host defense mechanisms during the tripartite virophage-giant virus-host cell interaction. We also present the latest advances regarding their origin, classification, and definition that have been widely discussed.

The Earth Is Small for "Leviathans": Long Distance Dispersal of Giant Viruses across Aquatic Environments.

Giant viruses of 'Megaviridae' have the ability to widely disperse around the globe. We herein examined 'Megaviridae' communities in four distinct aquatic environments (coastal and offshore seawater, brackish water, and hot spring freshwater), which are distantly located from each other (between 74 and 1,765 km), using a meta-barcoding method. We identified between 593 and 3,627 OTUs in each sample. Some OTUs were detected in all five samples tested as well as in many of the Tara Oceans metagenomes, suggesting the existence of viruses of this family in a wide range of habitats and the ability to circulate on the planet.

Giant Viruses-Big Surprises.

Viruses are the most prevalent infectious agents, populating almost every ecosystem on earth. Most viruses carry only a handful of genes supporting their replication and the production of capsids. It came as a great surprise in 2003 when the first giant virus was discovered and found to have a >1 Mbp genome encoding almost a thousand proteins. Following this first discovery, dozens of giant virus strains across several viral families have been reported. Here, we provide an updated quantitative and qualitative view on giant viruses and elaborate on their shared and variable features. We review the complexity of giant viral proteomes, which include functions traditionally associated only with cellular organisms. These unprecedented functions include components of the translation machinery, DNA maintenance, and metabolic enzymes. We discuss the possible underlying evolutionary processes and mechanisms that might have shaped the diversity of giant viruses and their genomes, highlighting their remarkable capacity to hijack genes and genomic sequences from their hosts and environments. This leads us to examine prominent theories regarding the origin of giant viruses. Finally, we present the emerging ecological view of giant viruses, found across widespread habitats and ecological systems, with respect to the environment and human health.